CN104830873B - A kind of thermophilic abnormal cocci IrrE albumen of site mutation and its application - Google Patents
A kind of thermophilic abnormal cocci IrrE albumen of site mutation and its application Download PDFInfo
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Abstract
The present invention has carried out the amino acid optimization of function motif with site mutation mode to the IrrE albumen and its homologous protein in thermophilic abnormal cocci Deinococcus geothermalis, is serine by second in functional domain motif 154LAELAR159 or the 5th alanine mutation.It is demonstrated experimentally that functional domain optimization can significantly improve the degeneration-resistant function of IrrE albumen and its homologous protein, the ultraviolet stress resistance effect for especially expressing it bacterial strain can improve more than 10 times.
Description
Technical field
The present invention relates to a kind of thermophilic abnormal cocci IrrE albumen of site mutation, and in particular to it is thermophilic that functional domain optimizes
Abnormal cocci Dgeo0395 homologous proteins, and the albuminoid is anti-to drying, oxidation and ultraviolet irradiation in enhancing prokaryotic hosts
Application in terms of property.
Background technology
IrrE is the important modulin of thermophilic abnormal cocci (Deinococcus geothermalis), by Dgeo0395
Gene code.According to the information of NCBI (US National Biotechnology Information center) database, the Dgeo0395 having now been found that is same
Source protein has 23.Known Dgeo0395 can specificly-response stress signal, improve the table that the bacterial strain resists gene in itself
Reach.
But have no the Dgeo0395 albumen in thermophilic abnormal cocci in the drying of enhancing other biological, oxidation and ultraviolet at present
Irradiate the research report of resistance function.Also have not seen and the key function motif of this albuminoid is analyzed, and it is prominent by fixed point
Change makes the report of function optimization.
The content of the invention
The purpose of the present invention is that the key function motif of Dgeo0395 gene coded proteins is optimized, and obtains regulation and control energy
The more preferable new protein sequence of power, to greatly improve the resistance of recombinant bacterial strain.
The present invention analyzes the domain of modulin Dgeo0395 by homologous modeling pattern.It was found that Dgeo0395 is by three
Domain is constituted, and wherein HTH domains are to participate in target gene promoter combination.Its HTH domain is by 3 α spirals (α
6th, α 7 and α 8) composition.
Contain important functional domain motif " 154LAELA159 " wherein on the spirals of α 7.23 in current ncbi database
Contain this section of functional domain motif sequence in the protein sequence of the homologous protein of Dgeo0395, and, this section of functional domain motif is only
It is found in abnormal cocci and belongs to (Deinococcus) bacterial strain (Fig. 1).
The method that the present invention utilizes Amino Acid-Induced Site-Directed Mutation, by degeneration-resistant experimental result, in this section of motif of com-parison and analysis
Influence of the different loci amino acid residue to the whole degeneration-resistant effects of modulin Dgeo0395, have found and thermophilic abnormal cocci is adjusted
The critical function domain motif 154LAELAR159 of control protein D geo0395 carries out the mutational formats of amino acid optimization.
Specific research work is as follows：
The amino acid optimization of the functional domain motif of Dgeo0395 genes
1) amino acid sequence (as shown in SEQ ID NO.2) to thermophilic abnormal cocci Dgeo0395 carries out structure domain-functionalities
Analysis.And to its important functional domain motif 154LAELAR159 (as shown in SEQ ID NO.3), carry out amino acid fixed point prominent
Become.
The following is Dgeo0395 amino acid sequences, wherein, underscore part is functional domain motif 154LAELAR159.
MTQGQTPPEELSADPSPETGALAPAKARMRELATAYARRLPGLDTHSLMSGLDATLTFMPMGDRDGAYDPEHRVVLI
NSRVRPERQRFTLAHEISHALLLGDDDLLSDLHDAYEGERLEQVIETLCNVGAAAILMPETLIDELLARFGPSGRA
RADVSASSALYALAERTSVPVLYAVCAVSRLEAESGEERLPEKALTVRASAGSPGVKYSLRPGTLIPDDHPVAVALE
TRLPITQESYVPFRSGRRMPAYVDAFPERQRVLVSFALLPKATKGGEQDESGV
The rite-directed mutagenesis is second alanine (155A) and/or the 5th third on functional domain motif 154LAELAR159
Propylhomoserin (158A) sports serine.
Wherein, deputy alanine mutation is serine, sequence such as SEQ ID in functional domain motif 154LAELAR159
Shown in NO.4；The alanine mutation of the 5th is serine, and sequence is as shown in SEQ ID NO.5.See the table below：
The amino acid optimization of the functional domain motif of table 1Dgeo0395 genes
| Sequence number | Sequence | Remarks |
| 3 | Leu Ala Glu Leu Ala Arg | 154LAELAR159 |
| 4 | Leu Ser Glu Leu Ala Arg | Second alanine mutation is serine |
| 5 | Leu Ala Glu Leu Ser Arg | 5th alanine mutation is serine |
2) recombinant strain of mutator is built, drying, oxidation and the ultraviolet irradiation stress of recombinant bacterial strain is identified
Resistance
This experiment shows：Functional domain motif 154LAELAR159 to the Function of thermophilic abnormal cocci Dgeo0395 very
It is important.Express the recombinant bacterium of thermophilic abnormal cocci Dgeo0395 optimizations albumen (Dgeo0395-A155S and Dgeo0395-A158S)
Compared with the recombinant bacterial strain before optimization, ultraviolet irradiation resistance is improved up to more than 10 times (see embodiment 3 and Fig. 4,5) for strain.
3) to thermophilic abnormal cocci Dgeo0395 homologous proteins DR-0167 (as shown in SEQ ID NO.6) and DGo_
CA2805 (as shown in SEQ ID NO.7) identical function domain motif carries out Amino Acid-Induced Site-Directed Mutation and ultraviolet irradiation stress resistance
Analysis.
This experiment shows：The functional domain motif function and modification scheme that the present invention is referred to are applicable not only to thermophilic abnormal cocci
Dgeo0395 albumen, is applied equally to numerous homologous proteins of Dgeo0395.Show functional domain motif transformation in homologous egg
The versatility acted in the outer degeneration-resistant sexual function of trillium.
Sequence table information
SEQ ID NO.1：The DNA sequence dna of Dgeo0395 genes；
SEQ ID NO.2：The amino acid sequence of Dgeo0395；
SEQ ID NO.3~5：It is shown in Table 1；
SEQ ID NO.6：The amino acid sequence of DR-0167；
SEQ ID NO.7：The amino acid sequence of DGo_CA2805.
Brief description of the drawings：
Fig. 1 Dego0395 and its homologous protein phylogenetic tree.Horizontal line mark is Dgeo0395 albumen, albumen sequence in figure
Row are included as all Dgeo0395 homologous proteins having now been found that, totally 23.Data from NCBI, (believe by US National biotechnology
Breath center) database.
Fig. 2 is the PCR primer containing thermophilic abnormal cocci Dgeo0395 sequences and carrier checking electrophoresis pattern；
Lane 1:2K plus Marker；Lane 2~3:PCR product of pJET-0395；Lane 4:
pRADZ3-0395/NdeI；Lane 5:pRADZ3-0395/NdeI+Spe I；Lane 6:pRADZ3-0395/Spe I；Lane
7:pRADZ3-0395/NdeI+Spe I
Before and after Fig. 3 ultraviolet irradiations, mitomycin C oxidation and drying stress impact, containing empty carrier bacterial strain (JM-Z3) and contain
The growing state for having the Escherichia coli (JM-0395) of the prokaryotic expression carrier of thermophilic abnormal cocci Dgeo0395 genes is contrasted；
Fig. 4 mitomycin Cs are aoxidized and drying stress impact is front and rear, the bacterial strain JM- containing functional domain motif mutational vector
Escherichia coli (the JM- of A155S and JM-A158S and the prokaryotic expression carrier containing thermophilic abnormal cocci Dgeo0395 genes
0395) growing state contrast；
Before and after the stress impact of Fig. 5 ultraviolet irradiations, bacterial strain JM-A155S and JM- containing functional domain motif mutational vector
The growth feelings of the Escherichia coli (JM-0395) of A158S and the prokaryotic expression carrier containing thermophilic abnormal cocci Dgeo0395 genes
Condition is contrasted.
Before and after the stress impact of Fig. 6 ultraviolet irradiations, bacterial strain JM-A155S and JM- containing functional domain motif mutational vector
A158S is bent with the existence of the Escherichia coli (JM-0395) of the prokaryotic expression carrier containing thermophilic abnormal cocci Dgeo0395 genes
Line.
Fig. 7 Dego0395 and its homologous protein sequence are compared and structural analysis.Frame area refers to functional domain motif for the present invention.
Protein sequence is included as all Dgeo0395 homologous proteins having now been found that, totally 15 in figure.Data come from NCBI (US Nationals
Biotechnology Information center) database.
Before and after the stress impact of Fig. 8 ultraviolet irradiations, the Escherichia coli (JM- of expression Deinococcus radiodurans DR-0167
DR0167) with the growing state contrast of the bacterial strain JM-A181S and JM-A184S of expressive function domain motif mutain.
Before and after the stress impact of Fig. 9 ultraviolet irradiations, the Escherichia coli (JM- of abnormal cocci DGO-CA2805 is penetrated in expression Gobi desert
CA2805) with the growing state contrast of the bacterial strain JM-S131A of expressive function domain motif mutain.
Specific embodiment
The plasmid lifted in following examples, bacterial strain are served only for being described in further detail the present invention, not to this hair
Bright substance is any limitation as.All unreceipted specific experiment conditions, it is according to routine well known to those skilled in the art
Condition, such as Sambrook equimoleculars are cloned：Laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) described in condition, or according to the condition proposed by manufacturer.
The plasmid lifted in embodiment, bacterium source are as follows：
Cloning vector pJET：It is ThermoFisher companies market products；
Shuttle plasmid pRADZ3：For this laboratory preserves；
Escherichia coli JM 109：It is Beijing Quan Shi King Companies commercially available prod.
Expression of the abnormal cocci Dgeo0395 gene orders in Escherichia coli that embodiment 1 is thermophilic
First, experimental technique
1. the Dgeo0395 gene orders design in the thermophilic genome of abnormal cocci strain DSM 11300 announced
1 pair of PCR specific primer：
0395-F：5′ACCACTAGT ATGACGCAGGGCCAGACCCC 3′
0395-R：5′ACCCATATG TCAGACACCCGACTCATCCT 3′
2. genes of interest sequence is amplified from the thermophilic genomic DNA of abnormal cocci strain DSM 11300 by PCR method
Row.
Reaction condition：94 DEG C of 10min, [94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 1.5min] 35 circulations, 72 DEG C
10min。
After 3.PCR products are through glue reclaim, it is cloned on carrier pJET, is named as pJET-0395, and sequence verification；Then
Dgeo0395 genes containing cohesive end are obtained by SpeI/NdeI double digestions and the pRADZ3 containing groEL promoters is carried
Body, Dgeo0395 genes are connected on pRADZ3 carriers, build coli expression carrier pRADZ3-0395.
4. the expression vector is converted into e. coli jm109, through PCR, digestion, sequence verification insetion sequence is correct (see figure
2,3) it is, JM-0395 by the Strain Designation.E.coli JM109 containing pRADZ3 empty plasmids are named as JM-Z3.
2nd, experimental result
Successfully construct the recombination bacillus coli engineered strain of expression Dgeo0395.
Stress resistance of the embodiment 2 containing thermophilic abnormal cocci bacterial strain Dgeo0395 genetic recombination engineered strains is tested
First, experiment material
Recombinant strain：The JM-0395 bacterial strains containing thermophilic abnormal cocci bacterial strain Dgeo0395 that embodiment 1 is obtained
Control strain：JM-Z3 bacterial strains containing empty plasmid described in embodiment 1.
2nd, experimental technique
1. by control strain and recombinant strain in the flat lining out activation of LB solid mediums；
2. picking single bacterium colony is inoculated in the LB liquid medium for being added with corresponding antibiotic, after 37 DEG C are cultivated into indexes
Phase；
3. 6,000rpm is centrifuged 5min collects thallines at room temperature, then will with the phosphate buffer (pH 7.0) of same volume
Thalline is washed twice, and concussion is uniform；
Followed by ultraviolet irradiation resistance, mitomycin C resistance and dry resistance assay
A. ultraviolet irradiation (UV) Analysis of Resistance：
1) bacterium solution is divided into isometric two parts；
2) collects thalline after a centrifugations of 6,000rpm at room temperature 5min, as control；
3) after taking another bacterium solution through ultraviolet irradiation 5min, 6,000rpm is centrifuged collects thalline after 5min at room temperature；
4) bacterium solution of different time sections is taken, 10 times are diluted to 10-5, take 10 μ L points on LB solid medium flat boards, 37 DEG C
Culture 2d, observation records the growing state of different strains；
5) bacterium solution of different time sections is taken, 10 times are diluted to 10-5, take 200 μ L coating LB solid medium flat boards, 37 DEG C of trainings
2d is supported, colony counts are recorded, bacterial strain survival rate is calculated, experiment is repeated 3 times.
B. mitomycin C aoxidizes Analysis of Resistance：
1) bacterium solution is divided into isometric two parts；
2) collects thalline after a centrifugations of 6,000rpm at room temperature 5min, as control；
3) thalline is resuspended in the LB fluid nutrient mediums of the mitomycin C of final concentration of 10 μ g/mL, 30 DEG C, 220rpm
Shaking table culture 5min, 10min, 15min；
4) bacterium solution of different time sections is taken, 10 times are diluted to 10-5, take 10 μ L points on LB solid medium flat boards, 37 DEG C
Culture 2d, observation records the growing state of different strains；
5) bacterium solution of different time sections is taken, 10 times are diluted to 10-5, take 200 μ L coating LB solid medium flat boards, 37 DEG C of trainings
2d is supported, colony counts are recorded, bacterial strain survival rate is calculated, experiment is repeated 3 times.
C. Analysis of Resistance is dried：
1) bacterium solution is divided into isometric two parts；
2) collects thalline after a centrifugations of 6,000rpm at room temperature 5min, as control；
3) another is sub-packed in the EP pipes of opening, is placed in aseptic drier (using particle silica gel as drier)；
4) a several different strains for batch are taken out every 10d, doubling dilution takes 10 μ L points and trained in LB solids to 10-5
Support on base flat board, 37 DEG C of culture 2d, observation records the growing state of different strains；
5) bacterium solution of different time points is taken, 10 times are diluted to 10-5, takes 200 μ L coating LB solid medium flat boards, 37 DEG C
Culture 2d, records colony counts, calculates bacterial strain survival rate, and experiment is repeated 3 times.
3rd, experimental result
As Fig. 3 shows, before the impact of ultraviolet irradiation, mitomycin C and drying stress, contain thermophilic abnormal cocci bacterial strain
The JM-0395 bacterial strains of Dgeo0395 are basically identical with the JM-Z3 strain growth states containing empty plasmid；
After the impact of ultraviolet irradiation, mitomycin C and drying stress, contain thermophilic abnormal cocci bacterial strain Dgeo0395 genes
JM-0395 recombinant bacterial strains upgrowth situation is good, and bacterium colony significantly number is more than the JM-Z3 bacterial strains containing only empty plasmid (see Fig. 5).It is wherein purple
External irradiation resistance and drying stress resistance improve 2 orders of magnitude, about 100 times compared with control strain；Mitomycin C oxidative stress resistance
Improve 3 orders of magnitude, about 1000 times.
Survival rate after bacterial strain is coerced is calculated and can clearly found：
Control strain survival rate is 0.644% ± 0.052% after UV irradiation；The JM-0395 bacterium of expression bacterial strain Dgeo0395
Strain survival rate is 45.570% ± 3.797%, improves nearly 70 times than control strain.
Control strain survival rate is 3.040% ± 0.929% after drought stress treatment, and JM-0395 Strain survival rates are
88.889% ± 7.274%, improve nearly 30 times than control strain.
Control strain is nearly all dead after mitomycin C oxidative stress, and JM-0395 Strain survival rates are
58.642% ± 4.660%, bacterial strain survival ability is significantly higher than control strain.
Before and after table 2 is ultraviolet irradiation, mitomycin C oxidation and drying stress impact, containing empty carrier bacterial strain (JM-Z3) and
Recombinant escherichia coli strain (JM-0395) survival rate containing thermophilic abnormal cocci Dgeo0395 genes compares.
Strain survival rate compares after 2 three kinds of Stress treatments of table
4th, experiment conclusion
Thermophilic abnormal cocci bacterial strain Dgeo0395 genes significantly enhance prokaryotes resistance ultraviolet radioactive, oxidation and dry
The ability of stress.
The amino acid optimization of the functional domain motif of embodiment 3 is thermophilic abnormal cocci Dgeo0395 genes builds
Analyze data is compared according to Homologous gene sequences, to the critical function of thermophilic abnormal cocci Dgeo0395 expressing proteins
Domain motif 154LAELAR159 has carried out amino acid sequence optimization.
First, experimental technique
Critical function domain motif 154LAELAR159 to thermophilic abnormal cocci modulin Dgeo0395 has carried out amino
Acid optimization.The domain of modulin Dgeo0395 is analyzed by homologous modeling pattern.Dgeo0395 is made up of three domains,
Wherein HTH domains are to participate in target gene promoter combination.Its HTH domain is by 3 α spirals (α 6, α 7 and α 8) group
Into containing important functional domain motif 154LAELA159 wherein on the spirals of α 7.Dgeo0395's is homologous in current ncbi database
Albumen finds 23 sequences altogether, is only found in abnormal cocci and belongs to (Deinococcus) bacterial strain (Fig. 1).In these protein sequences
Contain this section of functional domain motif.Using the method for Amino Acid-Induced Site-Directed Mutation, the different loci amino acid analyzed in this section of motif is residual
Influence of the base to the whole degeneration-resistant effects of modulin Dgeo0395.
1. to the amino acid sequence of thermophilic abnormal cocci Dgeo0395 is carried out into work(by way of Amino Acid-Induced Site-Directed Mutation
Energy domain motif 154LAELAR159 optimizes analysis.Using the method for fusion DNA vaccine by the nucleosides of target site coded amino acid
Acid sequence is mutated, and then obtains the protein mutant for changing site mutation.The mutational site of selection be 154L, 155A, 157L,
158A and 159R.154 leucines are sported into valine respectively, 155 alanine mutations are serine, and 157 is leucine
Valine is sported, 158 alanine mutations are serine, and 159 arginine sport lysine.
Primer sequence is as follows：
a-0395-F：5′accactagtatgacgcagggccagacccc 3′
d-0395-R：5′acccatatgtcagacacccgactcatcct 3′
b1-L154V-F:5′gggcgtgcgGTGgctgagctggcgcggcgggcagacgtga 3′
c1-L154V-R:5′tcacgtctgcccgccgcgccagctcagcCACcgcacgccc 3′
b2-A155S-F:5′gggcgtgcgctgAGCgagctggcgcggcgggcagacgtga 3′
c2-A155S-R:5′tcacgtctgcccgccgcgccagctcGCTcaccgcacgccc 3′
b3-L157V-F:5′gggcgtgcgctggctgagGTGgcgcggcgggcagacgtga 3′
c3-L157V-R:5′tcacgtctgcccgccgcgccagCACagccaccgcacgccc 3′
b4-A158S-F:5′gggcgtgcgctggctgagctgAGCcggcgggcagacgtga 3′
c4-A158S-R:5′tcacgtctgcccgcccGCTcagctcagccaccgcacgccc 3′
b5-R159K-F:5′gggcgtgcgctggctgagctggcgAAGcgggcagacgtga 3′
c5-R159K-R:5′tcacgtctgcccgCTTcgccagctcagccagcgcacgccc 3′
2. two primer b and c comprising rite-directed mutagenesis sequence are designed at nucleic acid sequences to be mutated, while designing the base
The upstream and downstream primer a and d of cause.With genome it is respectively that template PCR amplifications obtain two fragments 1,2 with primer a, b and c, d, so
Afterwards by 1,2 liang of fragments in molar ratio 1:As template after 1 mixing, the two fragments are merged with primer a, d.
Fragment 1,2 reaction conditions：94 DEG C of 10min, [94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 1.5min] 30 circulations, 72
℃10min。
Fusion reaction condition：94 DEG C of 10min, [94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 2min] 40 circulations, 72 DEG C
10min。
3. the recombination mutation fragment of acquisition is connected into carrier pRADZ3 and carry out Escherichia coli conversion (method reference implementation example
1) recombinant strain JM-L154V, JM-A155S, JM-L157V, JM-A158S-F, JM-R159K, are obtained.
2nd, experimental result
Successfully construct the critical function domain motif for expressing thermophilic abnormal cocci modulin Dgeo0395
Recombinant strain JM-L154V, JM-A155S, JM-L157V, JM- of 154LAELAR159 amino acid sites mutation
A158S-F、JM-R159K。
Embodiment 4 expresses thermophilic abnormal cocci bacterial strain Dgeo0395 albumen critical functions domain motif 154LAELAR159 optimizations
The stress resistance experiment of recombinant strain
First, experiment purpose
Identify each recombinant bacterial strain drying, oxidation and ultraviolet irradiation stress resistance, and with blank and the bacterium that sets out
Strain is compared.
2nd, experiment material
Recombinant strain：The critical function of the thermophilic abnormal cocci modulin Dgeo0395 of expression that embodiment 3 is obtained
The recombinant strain of domain motif 154LAELAR159 amino acid sites mutation：JM-L154V、JM-A155S、JM-L157V、
JM-A158S、JM-R159K。
Control strain：
JM-Z3 bacterial strains containing empty plasmid described in embodiment 1；
JM-0395 bacterial strains containing thermophilic abnormal cocci bacterial strain Dgeo0395 described in embodiment 1.
3rd, experimental technique
Each bacterial strain is tested to the experimental technique that drying, oxidation and ultraviolet irradiate three kinds of resistances of stress according to embodiment 2
Method is carried out.
4th, experimental result
1st, ultraviolet (UV) is coerced
Result see the table below
Each Strain survival rate compares under the ultraviolet of table 2 irradiation stress
During UV Stress treatments 10 minutes, control strain JM-Z3 is all dead, expresses thermophilic abnormal cocci Dgeo0395
The survival rate of genetic recombination bacterial strain JM-0395 drops to 0.125%, and expresses the recombinant bacterial strain JM- of Dgeo0395 optimization genes
The survival rate of A155S is 1.263%, expresses the survival rate of recombinant bacterial strain JM-A158S of Dgeo0395 optimization genes preferably, still
It is 62.5%.
During UV Stress treatments 15 minutes, the survival of thermophilic abnormal cocci Dgeo0395 genetic recombination bacterial strains JM-0395 is expressed
Rate is further reduced to 0.014%, and the survival rate for expressing the recombinant bacterial strain JM-A155S of Dgeo0395 optimization genes is
0.197%, the survival rate for expressing the recombinant bacterial strain JM-A158S of Dgeo0395 optimization genes is maintained at 12% or so (table 2).
UV irradiation survivorship curves show that the alanine mutation of 155 is serine in functional domain motif 154LAELAR159,
As shown in SEQ ID NO.4, make survival rate of the recombinant bacterial strain when irradiating 15 minutes than improving about 15 times before mutation；And 158
Alanine mutation be serine, as shown in SEQ ID NO.5 so that recombinant bacterial strain irradiation 15 minutes survival rate than mutation
It is preceding to improve nearly 900 times (table 2, Fig. 6).
2nd, the drying of recombinant bacterial strain JM-A155S and JM-A158S and oxidative stress resistance
The drying of recombinant bacterial strain JM-A155S and JM-A158S and oxidative stress resistance recombinant bacterial strain JM-0395 preceding with mutation
Unanimously (see Fig. 4).
5th, experiment conclusion
1st, the recombinant bacterial strain JM-A155S and JM-A158S of functional domain motif optimization is significantly high to ultraviolet spoke stress resistance
In starting strain JM-0395, more than 10 times (Fig. 5) is improved.Hence it is demonstrated that in 154LAELAR159,155 and 158 the third ammonia
Acid is the avtive spot for improving uvioresistant spoke stress function.
2nd, recombinant bacterial strain JM-L154V, JM-L157V and JM-R159K is to drying, oxidation and ultraviolet irradiation stress resistance
It is consistent with bacterial strain JM-0395, it is above empty plasmid bacterial strain.Prove, in 154LAELAR159, L154V, L157V and R159K site
Mutation do not influence to set out the degeneration-resistant activity of bacterium, i.e. the regulating and controlling effect on Dgeo0395 does not influence.
3rd, there is the functional domain motif 154LAELAR159 of thermophilic abnormal cocci Dgeo0395 raising protein D geo0395 to resist
The important function of reverse function.
The amino acid optimization transformation of IrrE homologous proteins functional domain motif and purple in the Deinococcus radiodurans of embodiment 5
Outer resistance assay identification
First, experiment purpose
Dgeo0395 homologous protein sequences are compared (see Fig. 1,7) using ncbi database gene information.
Homologous proteins of the Dgeo0395 in Deinococcus radiodurans (Deinococcus radiodurans) is DR0167, the albumen
The positions of α 7 equally contain present invention discover that critical function domain motif 180LAELAR185, amino acid sequence and Dgeo0395 phases
With (see Fig. 7).To in the critical function domain motif 180LAELAR185 of the homologous protein DR0167 in Deinococcus radiodurans
Two or the 5th alanine optimize mutation transformation, identify the functional domain motif in the ultraviolet resistance work(of recombination mutation albumen
Effect in energy.
2nd, experimental technique
1. to the amino acid of Deinococcus radiodurans IrrE homologous proteins DR0167 by way of Amino Acid-Induced Site-Directed Mutation
Sequence carries out space structure same position functional domain motif 180LAELAR185 and optimizes analysis.Also with fusion DNA vaccine
Be mutated for the nucleotide sequence of target site coded amino acid by method, and then obtains the protein mutant for changing site mutation.
It is two alanine in functional domain motif, 181A and 184A that the mutational site of selection is.It is by 181 alanine mutations respectively
Serine, 184 alanine mutations are serine.
Primer sequence is as follows：
a-dr0167-F：5′taactagtgtgcccagtgccaacgtcag 3′
d-dr0167-R：5′tacatatgtcactgtgcagcgtcct 3′
b1-A181S-F:5′ggccccaccgggcgcgccctcAGCgaactcgccaagcgggcc 3′
c1-A181S-R:5′ggcccgcttggcgagttcGCTgagggcgcgcccggtggggcc 3′
b2-A184S-F:5′ggccccaccgggcgcgccctcgccgaactcAGCaagcgggcc 3′
c2-A184S-R:5′ggcccgcttGCTgagttcggcgagggcgcgcccggtggggcc 3′
2. the Deinococcus radiodurans DR0167 original genes fragment and recombination mutation piece for obtaining, and section connection carrier
PRADZ3 simultaneously carries out Escherichia coli conversion (method reference implementation example 1,3), obtains recombinant strain JM-DR0167, JM-
A181S and JM-A184S.
3. the resistance assay method that each bacterial strain is irradiated to ultraviolet is carried out with reference to the experimental technique of embodiment 2.
3rd, experimental result
UV irradiation Stress treatments show that Deinococcus radiodurans DR-0167 can equally improve prokaryotic micro-organisms UV stress
Resistance.Equally, second in its functional domain motif 180LAELAR185 or the 5th alanine are optimized and sports an ammonia
Acid, further increases the ability (Fig. 8) that recombinant protein improves cell anti-ultraviolet radiation.Experimental result shows, functional domain motif
In 180LAELAR185 second alanine mutation for serine be former DR-0167 albumen protective effect improve only 10 times；And
The 5th alanine mutation proposes the protective effect of original DR-0167 albumen for serine in functional domain motif 180LAELAR185
2 orders of magnitude high, nearly 100 times (see Fig. 8).
4th, experiment conclusion
The DR-0167 of Deinococcus radiodurans is the homologous protein of Dgeo0395.It is same in DR-0167 to be sent out containing the present invention
Existing critical function domain motif, is 180LAELAR185.Sequence of amino acid is identical with Dgeo0395.Experimental result confirmation,
Second in its functional domain motif 180LAELAR185 or the 5th alanine are optimized and sport serine, is further carried
Recombinant protein high improves the ability of cell anti-ultraviolet radiation.Test result indicate that, the Dgeo0395's that the present invention is referred to is important
Second or the 5th alanine mutation are serine in functional domain motif, improve the protein protection prokaryotic UV stress resistances
Discovery, be equally applicable to the degeneration-resistant function modificationses to Dgeo0395 homologous proteins.
The amino acid sequence transformation of IrrE homologous proteins functional domain motif and ultraviolet resistance in the Gobi abnormal cocci of embodiment 6
Experimental identification
First, experiment purpose
Dgeo0395 homologous protein sequences are compared (see Fig. 1,7) using ncbi database gene information.
Homologous proteins of the Dgeo0395 in Gobi abnormal cocci (Deinococcus gobiensis) is DGo_CA2805, the albumen
The positions of α 7 equally contain present invention discover that critical function domain motif 127LAELSR132, amino acid sequence and Dgeo0395 are not
Together, its 5th is not alanine, but serine (see Fig. 7).To the homologous protein DGo_CA2805 in Gobi abnormal cocci
The 5th serine of critical function domain motif 127LAELSR132 carry out inverse transition and transform alanine as, identify the function
Effect of the domain motif in the ultraviolet degeneration-resistant sexual function of recombination mutation albumen.
2nd, experimental technique
1. to the amino of Gobi abnormal cocci IrrE homologous proteins DGo_CA2805 by way of Amino Acid-Induced Site-Directed Mutation
Acid sequence carries out space structure same position functional domain motif 127LAELSR132 and is analyzed.Also with the side of fusion DNA vaccine
Be mutated for the nucleotide sequence of target site coded amino acid by method, and then obtains the protein mutant for changing site mutation.Choosing
It is the 5th serine in functional domain motif that the mutational site for taking is.It is alanine by 131 serine inverse transitions.
Primer sequence is as follows：
a-CA2805-F：5′taactagtatgcgcgagctggcggcgg 3′
d-CA2805-R：5′tacatatggtgagagggagacgcgct 3′
b1-S131A-F:5′cgcgccctggccgagttgGCCcgccgcgccgacgtgagt 3′
c1-S131A-R:5′actcacgtcggcgcggcgGGCcaactcggccagggcgcg 3′
2. the Gobi abnormal cocci DGo_CA2805 original genes fragment and recombination mutation piece for obtaining, and section connection carrier
PRADZ3 simultaneously carries out Escherichia coli conversion (method reference implementation example 1,3), obtains recombinant strain JM-CA2805, JM-
S131A。
3. the resistance assay method that each bacterial strain is irradiated to ultraviolet is carried out with reference to the experimental technique of embodiment 2.
3rd, experimental result
UV irradiation Stress treatments show that Gobi abnormal cocci DGo_CA2805 can equally improve prokaryotic micro-organisms UV stress
Resistance.Equally, it is alanine to the 5th mutant serine of its functional domain motif 127LAELSR132, significantly reduces restructuring egg
The white ability (Fig. 9) for improving cell anti-ultraviolet radiation.Experimental result shows, the 5th silk in functional domain motif 127LAELSR132
Histidine mutations are that the protective effect of original DGo_CA2805 albumen is reduced by 2 orders of magnitude, about 100 times (see Fig. 9) by alanine.
4th, experiment conclusion
The DGo_CA2805 of Gobi abnormal cocci is the homologous protein of Dgeo0395.Equally containing this hair in DGo_CA2805
The critical function domain motif of bright discovery, is 127LAELSR132.Sequence of amino acid is different from Dgeo0395, its 5th ammonia
Base acid is naturally serine.This site amino acids is carried out inverse transition by the present embodiment, and verify the functional domain motif
Effect in the ultraviolet degeneration-resistant sexual function of recombination mutation albumen.Experimental result is confirmed, in its functional domain motif 127LAELSR132
5th serine carries out inverse transition for alanine, hence it is evident that reduce the ability that recombinant protein improves cell anti-ultraviolet radiation.
Experimental result proves from opposite direction, second or the 5th the third ammonia in the critical function domain motif of the Dgeo0395 that the present invention is referred to
Acid mutation is serine, improves the discovery of the protein protection prokaryotic UV stress resistances, is equally applicable to same to Dgeo0395
The degeneration-resistant function modificationses of source protein.
Claims (2)
1. the functional domain base in thermophilic abnormal cocci (Deinococcus geothermalis) IrrE albumen and its homologous protein
Applications of the sequence 154LAELAR159 in degeneration-resistant function modificationses, the nucleotide sequence such as SEQ ID of the 154LAELAR159
Shown in NO.3；
It by the second alanine of amino acid sequence shown in SEQ ID NO.3 or the 5th alanine mutation is silk that the transformation is
Propylhomoserin.
2. the application described in claim 1, it is described it is degeneration-resistant be uvioresistant irradiation stress.
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| CN201510236790.8A CN104830873B (en) | 2015-05-11 | 2015-05-11 | A kind of thermophilic abnormal cocci IrrE albumen of site mutation and its application |
| AU2016260738A AU2016260738B2 (en) | 2015-05-11 | 2016-05-09 | IrrE protein functional domain for improving anti-oxidation capability of cell and application thereof |
| PCT/CN2016/081403 WO2016180300A1 (en) | 2015-05-11 | 2016-05-09 | Irre protein functional domain for improving anti-oxidation capability of cell and application thereof |
| US15/553,599 US10738314B2 (en) | 2015-05-11 | 2016-05-09 | IrrE protein functional domain for improving anti-oxidation capability of cell and application thereof |
| CA2977853A CA2977853C (en) | 2015-05-11 | 2016-05-09 | Irre protein functional domain for improving anti-oxidation capability of cell and application thereof |
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| CN109097374A (en) * | 2018-07-24 | 2018-12-28 | 天津科技大学 | A kind of preparation method, bacterial strain and its application of pseudomonas aeruginosa engineered strain |
| CN109055417B (en) * | 2018-08-28 | 2020-07-07 | 浙江新和成股份有限公司 | Recombinant microorganism, preparation method thereof and application thereof in production of coenzyme Q10 |
| CN109182310B (en) * | 2018-09-26 | 2021-10-08 | 中国农业科学院生物技术研究所 | Application of radiation-resistant Gobi deinococcus keratin gene |
| CN109402081B (en) * | 2018-11-27 | 2021-08-20 | 江南大学 | Amylosucrase mutant and preparation method and application thereof |
| CN113151293B (en) * | 2020-10-20 | 2023-03-10 | 中国农业科学院生物技术研究所 | Stress-resistant gene line AcDwEm and application thereof in improving salt resistance, drought resistance and high temperature resistance of crops |
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| CN101591630A (en) * | 2009-06-30 | 2009-12-02 | 新疆农业科学院微生物应用研究所 | The microorganism anti-ultraviolet radiation preparation of strange coccus of a kind of radiation hardness and generation |
| CN101671679A (en) * | 2009-04-30 | 2010-03-17 | 浙江大学 | Resistance-relevant gene and applications of radiation-resistant cocci |
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| CN101696414B (en) * | 2009-11-10 | 2012-01-25 | 中国农业科学院生物技术研究所 | Gene capable of improving radiation resistance of organisms and application thereof |
| CN202443018U (en) * | 2012-03-05 | 2012-09-19 | 中国农业科学院生物技术研究所 | Gold-labeled kit for rapid joint detection of salt-resistant HIN protein and IrrE protein |
| WO2014072517A1 (en) * | 2012-11-12 | 2014-05-15 | Institució Catalana De Recerca I Estudis Avançats | Methods and kits for the prognosis of colorectal cancer |
| CN104830873B (en) * | 2015-05-11 | 2017-06-30 | 中国农业科学院生物技术研究所 | A kind of thermophilic abnormal cocci IrrE albumen of site mutation and its application |
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| CN101671679A (en) * | 2009-04-30 | 2010-03-17 | 浙江大学 | Resistance-relevant gene and applications of radiation-resistant cocci |
| CN101591630A (en) * | 2009-06-30 | 2009-12-02 | 新疆农业科学院微生物应用研究所 | The microorganism anti-ultraviolet radiation preparation of strange coccus of a kind of radiation hardness and generation |
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| US10738314B2 (en) | 2020-08-11 |
| CN104830873A (en) | 2015-08-12 |
| US20180163215A1 (en) | 2018-06-14 |
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